[Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin].

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

A new metabolic pathway for sym-homospermidine synthesis in an extreme thermophile, Thermus thermophilus.

In an extreme thermophile, Thermus thermophilus, sym-homospermidine is synthesized by the actions of two enzymes. The first enzyme coded by dhs gene (annotated to be deoxyhypusine synthase gene) catalyzes synthesis of an intermediate, supposed to be 1,9-bis(guanidino)-5-aza-nonane (=N1, N11-bis(amidino)-sym-homospermidine), from two molecules of agmatine in the presence of NAD. The second enzyme (aminopropylagmatinase) coded by speB gene catalyzes hydrolysis of the intermediate compound to sym-homospermidine releasing two molecules of urea.

Profiling of lipids in Thermus thermophilus HB8 grown under various conditions.

The membrane lipids of Thermus species have unique structures. Only four polar lipid species have so far been identified in Thermus thermophilus HB8; namely, are two phosphoglycolipids and two glycolipids, both of which have three branched fatty acid chains. Other lipid molecules may be present; however, they have not been identified so far. To clarify the whole lipid profile of T. thermophilus HB8, we cultured this organism under four different growth (temperature and/or nutrition) conditions and analyzed the compositions of polar lipids and fatty acids by high-performance thin-layer chromatography (HPTLC) and gas chromatograph-mass spectrometry (GCï½°MS), respectively. Thirty-one lipid spots were detected on HPTLC plates and profiled in terms of the presence or absence of phosphate, amino, and sugar groups. Then, we allocated ID numbers to all the spots. Comparative analyses of these polar lipids showed that the diversity of lipid molecules increased under high temperature and minimal medium conditions. In particular, aminolipid species increased under high temperature conditions. As for the fatty acid comparison by GC-MS, iso-branched even-numbered carbon atoms, which are unusual in this organism, significantly increased under the minimal medium condition, suggesting that kinds of branched amino acids at the fatty acid terminus varies under different nutrition conditions. In this study, several unidentified lipids were detected, and elucidation of the lipid structures will provide important information on the environmental adaptation of bacteria.

Whole genome analyses for c-type cytochromes associated with respiratory chains in the extreme thermophile, Thermus thermophilus.

In thermophilic microorganisms, c-type cytochrome (cyt) proteins mainly function in the respiratory chain as electron carriers. Genome analyses at the beginning of this century revealed a variety of genes harboring the heme c motif. Here, we describe the results of surveying genes with the heme c motif, CxxCH, in a genome database comprising four strains of Thermus thermophilus, including strain HB8, and the confirmation of 19 c-type cytochromes among 27 selected genes. We analyzed the 19 genes, including the expression of four, by a bioinformatics approach to elucidate their individual attributes. One of the approaches included an analysis based on the secondary structure alignment pattern between the heme c motif and the 6th ligand. The predicted structures revealed many cyt c domains with fewer ß-strands, such as mitochondrial cyt c, in addition to the ß-strand unique to Thermus inserted in cyt c domains, as in T. thermophilus cyt c552 and caa3 cyt c oxidase subunit IIc. The surveyed thermophiles harbor potential proteins with a variety of cyt c folds. The gene analyses led to the development of an index for the classification of cyt c domains. Based on these results, we propose names for T. thermophilus genes harboring the cyt c fold.

ThermusQ: Toward the cell simulation platform for Thermus thermophilus.

ThermusQ is a website (https://www.thermusq.net/) that aims to gather all the molecular information on Thermus thermophilus and to provide a platform to easily access the whole view of the bacterium. ThermusQ comprises the genome sequences of 22 strains from T. thermophilus and T. oshimai strains, plus the sequences of known Thermus phages. ThermusQ also contains information and map diagrams of pathways unique to Thermus strains. The website provides tools to retrieve sequence data in different ways. By gathering the whole data of T. thermophilus strains, the strainspecific characteristics was found. This bird's-eye view of the whole data will lead the research community to identify missing important data and the integration will provide a platform to conduct future biochemical simulations of the bacterium.

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19 Šresolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Šresolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Description of five novel thermophilic species of the genus Thermus: Thermus hydrothermalis sp. nov., Thermus neutrinimicus sp. nov., Thermus thalpophilus sp. nov., Thermus albus sp. nov., and Thermus altitudinis sp. nov., isolated from hot spring sediments.

Biological denitrification is a significant process in nitrogen biogeochemical cycle of terrestrial geothermal environments, and Thermus species have been shown to be crucial heterotrophic denitrifier in hydrothermal system. Five Gram-stain negative, aerobic and rod-shaped thermophilic bacterial strains were isolated from hot spring sediments in Tibet, China. Phylogenetic analysis based on 16S rRNA gene and whole genome sequences indicated that these isolates should be assigned to the genus Thermus and were most closely related to Thermus caldifontis YIM 73026T, and Thermus brockianus YS38T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the five strains and the type strains of the genus Thermus were lower than the threshold values (95% and 70%, respectively) recommended for bacterial species, which clearly distinguished the five isolates from other species of the genus Thermus and indicated that they represent independent species. Colonies are circular, convex, non-transparent. Cell growth occurred at 37-80 °C (optimum, 60-65 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-2.0% (w/v) NaCl (optimum, 0-0.5%). Denitrification genes (narG, nirK, nirS, and norB genes) detected in their genomes indicated their potential function in nitrogen metabolism. The obtained results combined with those of morphological, physiological, and chemotaxonomic characteristics, including the menaquinones, polar lipids, and cellular fatty acids showed that the isolates are proposed as representing five novel species of the genus Thermus, which are proposed as Thermus hydrothermalis sp. nov. SYSU G00291T, Thermus neutrinimicus sp. nov. SYSU G00388T, Thermus thalpophilus sp. nov. SYSU G00506T, Thermus albus sp. nov. SYSU G00608T, Thermus altitudinis sp. nov. SYSU G00630T.

Enhancement of PET degradation by PET depolymerase with the microbe addition.

The degradation of polyethylene terephthalate (PET) by modified PET depolymerase has recently attracted much attention. We found that mixing a PET depolymerase with non-genetically modified Thermus sp. can enhance its PET-degrading activity by 7.7-fold. This approach is attractive for constructing a sustainable PET recycling system.

Incomplete denitrification phenotypes in diverse Thermus species from diverse geothermal spring sediments and adjacent soils in southwest China.

A few members of the bacterial genus Thermus have been shown to be incomplete denitrifiers, terminating with nitrite (NO2-) or nitrous oxide (N2O). However, the denitrification abilities of the genus as a whole remain poorly characterized. Here, we describe diverse denitrification phenotypes and genotypes of a collection of 24 strains representing ten species, all isolated from a variety of geothermal systems in China. Confirmed terminal products of nitrate reduction were nitrite or N2O, while nitric oxide (NO) was inferred as the terminal product in some strains. Most strains produced N2O; complete denitrification was not observed. Denitrification phenotypes were largely consistent with the presence of denitrification genes, and strains of the same species often had the same denitrification phenotypes and largely syntenous denitrification gene clusters. Genes for nirS and nirK coexisted in three Thermus brockianus and three Thermus oshimai genomes, which is a unique hallmark of some denitrifying Thermus strains and may be ecologically important. These results show that incomplete denitrification phenotypes are prominent, but variable, within and between Thermus species. The incomplete denitrification phenotypes described here suggest Thermus species may play important roles in consortial denitrification in high-temperature terrestrial biotopes where sufficient supply of oxidized inorganic nitrogen exists.

Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding.

G-quadruplexes (G4s) are unusual stable DNA structures that cause genomic instability. To overcome the potential barriers formed by G4s, cells have evolved different families of proteins that unfold G4s. Pif1 is a DNA helicase from superfamily 1 (SF1) conserved from bacteria to humans with high G4-unwinding activity. Here, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif1) complexed with a G4. Our structure reveals that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B which constitute a G4-Recognizing Surface (GRS). The overall structure of the G4 maintains its three-layered propeller-type G4 topology, without significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 are recognized by GRS residues mainly through electrostatic, ionic interactions, and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Compared with previously solved structures of SF2 helicases in complex with G4, our structure reveals how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.

An Argonaute from Thermus parvatiensis exhibits endonuclease activity mediated by 5' chemically modified DNA guides.

Prokaryotic Argonaute (pAgo) nucleases with precise DNA cleavage activity show great potential for gene manipulation. Extensive biochemical studies have revealed that recognition of guides with different 5' groups by Ago is important for biocatalysis. Here, we identified an Ago from the thermophilic Thermus parvatiensis ( TpsAgo) and analyzed the regulatory effect of 5'-modified guides on TpsAgo cleavage activity. Recombinant TpsAgo cleaves single-stranded DNA and RNA targets at 65-90°C, which is mediated by a 5' hydroxyl or phosphate DNA guide. Notably, TpsAgo can utilize various 5'-modified DNA guides for catalysis, including 5'-NH 2C 6, 5'-Biotin, 5'-FAM and 5'-SHC 6 guides. Moreover, TpsAgo performs programmable cleavage of double-stranded DNA at temperatures over 80°C and strongly tolerates NaCl concentrations up to 3.2 M. These results provide insight into the catalytic performance of Agos by guide regulation, which may facilitate their biotechnological applications.

Thermus brevis sp. nov., a moderately thermophilic bacterium isolated from a hot spring microbial mat.

Three closely related, facultative anaerobic, Gram-stain-negative, twitching motile, short rod-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05001T, SYSU G05003 and SYSU G05004, were isolated from a hot spring microbial mat, collected from Rehai National Park, Tengchong, Yunnan Province, south-western China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these three strains were closely related to Thermus scotoductus SE-1T (97.97, 98.18, 97.90 % sequence similarity). Whole genome sequencing and polyphasic taxonomic approach were used to determine the genomic profile and taxonomic status of the novel strain SYSU G05001T. Cell growth occurred at 37-80 °C (optimum, 55 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum, 1%). Thiosulfate enhanced cell growth. MK-8 was the predominant menaquinone. The major cellular fatty acids included iso-C15 : 0, iso-C17 : 0 and anteiso-C15 : 0. The major polar lipids were consisted of aminophospholipid, glycolipid and phospholipids. The whole genome of strain SYSU G05001T consisted of 2.55 Mbp and the DNA G+C content was 64.94 mol%. The average nucleotide identity (≤94.95 %) and digital DNA-DNA hybridization (≤62.3 %) values between strain SYSU G05001T and other members of the genus Thermus were all lower than the threshold values recommended for distinguishing novel prokaryotic species. On the basis of the presented polyphasic evidence and genotypic data, it is proposed that strain SYSU G05001T (=KCTC 82627T=MCCC 1K06118T) represents a novel species of the genus Thermus, for which the name Thermus brevis sp. nov. is proposed.

Enhancing Cohort PASA Efficiency from Lessons Assimilated by Mutant Genotyping in C. elegans.

Classical restriction fragment length polymorphism (RFLP) and sequencing are labor-intensive and expensive methods to study single base changes, whereas polymerase chain reaction amplification of specific alleles (PASA) or allele-specific polymerase chain reaction (ASPCR) is a PCR-based application that allows direct detection of any point mutation by analyzing the PCR products in an ethidium bromide-stained agarose or polyacrylamide gel. PASA is based on oligonucleotide primers containing one or more 3' mismatch with the target DNA making it refractory to primer extension by Thermus aquaticus DNA polymerase lacking the 3' to 5' exonuclease proofreading activity because of which it is also called amplification refractory mutation system-PCR (ARMS-PCR). This technique has found application in detection of allele, mutation, single-nucleotide polymorphisms (SNPs) causing genetic and infectious diseases. This chapter describes an approach of cohort PASA in context of genotyping single and double mutant worms generated to study the process of cell migration and axon outgrowth in C. elegans. Single worm-based cohort PASA allows genotyping for identification of single base mutations; particularly it is convenient method to detect mutations without a visible phenotype.

A Novel Trehalose Synthase for the Production of Trehalose and Trehalulose.

A novel putative trehalose synthase gene (treM) was identified from an extreme temperature thermal spring. The gene was expressed in Escherichia coli followed by purification of the protein (TreM). TreM exhibited the pH optima of 7.0 for trehalose and trehalulose production, although it was functional and stable in the pH range of 5.0 to 8.0. Temperature activity profiling revealed that TreM can catalyze trehalose biosynthesis in a wide range of temperatures, from 5°C to 80°C. The optimum activity for trehalose and trehalulose biosynthesis was observed at 45°C and 50°C, respectively. A catalytic reaction performed at the low temperature of 5°C yielded trehalose with significantly reduced by-product (glucose) production in the reaction. TreM displayed remarkable thermal stability at optimum temperatures, with only about 20% loss in the activity after heat (50°C) exposure for 24 h. The maximum bioconversion yield of 74% trehalose (at 5°C) and 90% trehalulose (at 50°C) was obtained from 100 mM maltose and 70 mM sucrose, respectively. TreM was demonstrated to catalyze trehalulose biosynthesis utilizing the low-cost feedstock jaggery, cane molasses, muscovado, and table sugar. IMPORTANCE Trehalose is a rare sugar of high importance in biological research, with its property to stabilize cell membrane and proteins and protect the organism from drought. It is instrumental in the cryopreservation of human cells, e.g., sperm and blood stem cells. It is also very useful in the food industry, especially in the preparation of frozen food products. Trehalose synthase is a glycosyl hydrolase 13 (GH13) family enzyme that has been reported from about 22 bacterial species so far. Of these enzymes, to date, only two have been demonstrated to catalyze the biosynthesis of both trehalose and trehalulose. We have investigated the metagenomic data of an extreme temperature thermal spring to discover a novel gene that encodes a trehalose synthase (TreM) with higher stability and dual transglycosylation activities of trehalose and trehalulose biosynthesis. This enzyme is capable of catalyzing the transformation of maltose to trehalose and sucrose to trehalulose in a wide pH and temperature range. The present investigation endorses the thermal aquatic habitat as a promising genetic resource for the biocatalysts with high potential in producing high-value rare sugars.

Enhancement of large ring cyclodextrin production using pretreated starch by glycogen debranching enzyme from Corynebacterium glutamicum.

Synthesis of large-ring cyclodextrins (LR-CDs) in any significant amount has been challenging. This study enhanced the LR-CDs production by Thermus filiformis amylomaltase (TfAM) enzyme by starch pretreatment using glycogen debranching enzyme from Corynebacterium glutamicum (CgGDE). CgGDE pretreated tapioca starch gave LR-CD conversion of 31.2 ± 2.2%, compared with LR-CDs produced from non-treated tapioca starch (16.0 ± 2.4%). CgGDE pretreatment enhanced amylose content by approximately 30%. Notably, a shorter incubation time of 1 h is sufficient for CgGDE starch pretreatment to produce high LR-CD yield, compared with 6 h required for the commercial isoamylase. High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD) and Gel Permeable Chromatography (GPC) revealed that CgGDE is more efficient than the commercial isoamylase in debranching tapioca starch and gave lower molecular weight products. In addition, lower amount of by-products (linear oligosaccharides) were detected in cyclization reaction when using CgGDE-pretreated starch. In conclusion, CgGDE is a highly effective enzyme to promote LR-CD synthesis from starch with a shorter incubation time than the commercial isoamylase.

Measuring the absolute abundance of the microbiome by adding yeast containing 16S rRNA gene from a hyperthermophile.

High-throughput sequencing (HTS) of 16S rRNA gene amplicons provides compositional information regarding the microbial community, but not the absolute abundance of the bacteria. We aimed to develop a standardized method for quantifying the absolute abundance of bacteria in microbiome studies. To demonstrate the utility of our approach, we quantified the number of bacteria from the compositional data of the fecal and cecal microbiomes. The 16S rRNA gene of a hyperthermophile, Thermus aquaticus, was cloned into Pichia pastoris (yeast) genome, and an equivalent amount of the yeast was added to the stool and cecal samples of mice before DNA extraction. 16S rRNA gene library construction and HTS were performed after DNA extraction. The absolute abundances of bacteria were calculated using T. aquaticus reads. The average relative abundances of T. aquaticus in the five stool and five cecal samples were 0.95% and 0.33%, respectively, indicating that the number of bacteria in a cecum sample is 2.9 times higher than that in a stool sample. The method proposed for quantifying the absolute abundance of the bacterial population in this study is expected to overcome the limitation of showing only compositional data in most microbiome studies.

Molecular basis for DarT ADP-ribosylation of a DNA base.

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.

Broken symmetry between RNA enantiomers in a crystal lattice.

Explaining the origin of the homochirality of biological molecules requires a mechanism of disrupting the natural equilibrium between enantiomers and amplifying the initial imbalance to significant levels. Authors of existing models have sought an explanation in the parity-breaking weak nuclear force, in some selectively acting external factor, or in random fluctuations that subsequently became amplified by an autocatalytic process. We have obtained crystals in which l- and d-enantiomers of short RNA duplexes assemble in an asymmetric manner. These enantiomers make different lattice contacts and have different exposures to water and metal ions present in the crystal. Apparently, asymmetry between enantiomers can arise upon their mutual interactions and then propagate via crystallization. Asymmetric racemic compounds are worth considering as possible factors in symmetry breaking and enantioenrichment that took place in the early biosphere.

Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation.

In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (MeAAs), is less efficient, especially when MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of MeAAs, achieving the incorporation of nine distinct MeAAs into both linear and thioether-macrocyclic peptide scaffolds.

Role of bacterial RNA polymerase gate opening dynamics in DNA loading and antibiotics inhibition elucidated by quasi-Markov State Model.

To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with σ factor) loads the promoter DNA via the flexible loading gate created by the clamp and ß-lobe, yet their roles in DNA loading have not been characterized. We used a quasi-Markov State Model (qMSM) built from extensive molecular dynamics simulations to elucidate the dynamics of Thermus aquaticus holoenzyme's gate opening. We showed that during gate opening, ß-lobe oscillates four orders of magnitude faster than the clamp, whose opening depends on the Switch 2's structure. Myxopyronin, an antibiotic that binds to Switch 2, was shown to undergo a conformational selection mechanism to inhibit clamp opening. Importantly, we reveal a critical but undiscovered role of ß-lobe, whose opening is sufficient for DNA loading even when the clamp is partially closed. These findings open the opportunity for the development of antibiotics targeting ß-lobe of RNAP. Finally, we have shown that our qMSMs, which encode non-Markovian dynamics based on the generalized master equation formalism, hold great potential to be widely applied to study biomolecular dynamics.